Otitis media (OM), or middle ear infection, is a highly prevalent pediatric disease worldwide, ranking first in reasons why children make physician's office and emergency room visits, undergo surgery requiring general anesthesia, and experience hearing loss. Wide use of broad-spectrum antibiotics to treat OM has been a major driving force behind the sobering global emergence of multiple antibiotic resistant strains of the three bacterial species that predominate in OM. Recent licensure of several capsular conjugate vaccines directed against Streptococcus pneumoniae (Spn) has caused the microbiology of OM to shift worldwide, with the relative proportion of cases due to our studied microorganism (nontypeable Haemophilus influenzae or NTHI) increasing significantly. NTHI is now considered equivalent to Spn as a causative agent of all cases of acute OM, yet remains the predominant pathogen of chronic OM, recurrent OM, and OM in children who fail antibiotic treatment. There is thus a pressing need to develop vaccines to prevent, or even possibly resolve existing OM due to NTHI. We have designed and extensively tested vaccine candidates for the prevention and resolution of NTHI-induced OM that are derived from two bacterial adhesins/virulence determinants: OMP P5 and the Type IV twitching pilus (or Tfp). These candidates have shown significant promise to date in pre-clinical efficacy trials; however there are, as yet, a few remaining gaps in the understanding required to complete their development. Thereby, herein, we propose three highly complementary and integrated specific aims to fill these final gaps. In Specific Aim 1, we will investigate in vitro how viral and bacterial co-infection, adherence to polarized respiratory tract epithelium as well as exposure to distinct micro-environmental cues (e.g. temperature, nutrient availability) that distinguish NTHI's commensal home (nasopharynx or NP) from the site of active disease (middle ear) influence vaccine antigen expression as these candidates are derived from adhesins integral to NTHI's ability to colonize and form biofilms. We will also determine the ability to prevent or disrupt adherence and biofilm formation using antibodies directed at these vaccine antigens under diverse conditions designed to better mimic those encountered in the human host. In Specific Aim 2, we will use chinchilla models to determine vaccine antigen expression and assess vaccine efficacy in vivo, under conditions of viral and bacterial co-infection. The natural progression of disease from NP colonization to overt ascending infection of the middle ear that occurs in these models will also allow us to assess concordance of the effect of diverse micro-environmental cues on vaccine antigen expression in vitro (Aim 1) versus in vivo. In Specific Aim 3, we will determine the relative boostability, durability, and breadth of protective efficacy afforded by two Tfp and OMP P5-derived vaccine candidates following SQ (traditional, parenteral) vs. transcutaneous immunization (TCI) (novel, needle-free), as well as characterize the induced correlates of immune protection. Successful conduct of the proposed studies will remove all final barriers to vaccine completion.